Since the time of Emil Fischer, it has been a goal of organic chemists to design and chemically synthesize proteins. In recent years there has been a growing interest in the de novo design of proteins, particularly helix-bundle proteins, and their production by means of chemical synthesis or recDNA-expression. Examples of helix-bundle proteins designed de novo are provided by M. Hecht et al. (Science, 1990, vol. 249, pages 884-891), by L. Regan et al. (Science, 1988, vol. 241, pages 976-978), by W. F. DeGrado et al. (Science, 1989, vol. 243, pages 622-628), and by N. E. Zhou et al. (Biochemistry, 1992, vol. 31, pages 5739-5746).
In some cases, unexpected results have been obtained in which the experimentally observed structure of synthetic helix bundles has been different than the intended structure because of the uncontrolled nature of non-covalent intermolecular association, e.g., see B. Lovejoy et al. (Science, 1993, vol. 259, pages 1288-1293). To avoid such problems, helix bundle proteins have been made by the preparation of covalent arrays linked through porphyrin molecules or through metal chelate complexes. Preliminary evidence indicates that the expected structures have been achieved, e.g., see T. Sasaki et al. (J. Am. Chem. Soc., 1989, vol. 111, pages 380-381) and R. M. Ghadiri et al. (J. Am. Chem. Soc., 1992, vol. 114, pages 4000-4002).
An alternative, earlier approach to the preparation of covalent peptide arrays of predetermined secondary and tertiary structure is the "template assembled synthetic protein" (TASP) concept, disclosed by M. Mutter (Peptides-Chemistry and Biology, Proceedings of the 10th American Peptide Symposium; Marshall, G. R., Ed.; Escom: Leiden, 1988; pages 349-353). A template molecule is used to covalently anchor arrays of secondary structural elements. The distinctive feature of the TASP approach is the nonlinear topology used; the molecule is made up of an array of branched polypeptides, rather than the folded linear polypeptide chain of natural proteins, e.g., M. Mutter et al., (Angew. Chem., Int. Ed. Engl., 1989, vol. 28, pages 535-554). It is anticipated that this elegant concept will have a profound effect on the de novo design of proteins.
However, conventional synthetic approaches for preparing TASP molecular assemblies are arduous and/or provide low yields. For example, both stepwise solid phase synthesis (SPPS) and protected segment condensation approaches have been employed with limited success. M. Mutter, M. et al. (J. Am. Chem. Soc., 1992, vol. 114, pages 1463-1470) discloses an example of a stepwise solid phase synthesis (SPPS) of a template assembled synthetic protein. In addition, B. Dorner et al. (Innovation and Perspectives in Solid Phase Synthesis, Roger Epton Ed.; Intercept Limited: Andover, 1992; pages 163-170) and by I. Ernest et al. (Tetrahedron Lett., 1990. vol. 31, pages 4015-4018) disclose examples of the protected segment condensation approach for synthesizing template assembled synthetic proteins. Only a minimal number of TASP molecules have been produced by arduous synthetic efforts, e.g. supra and M. Mutter et al. (Proteins, 1989, vol. 5, pages 13-21) and G. Tuchscherer (Protein Science, 1992, vol. 1, pages 1377-1386). Despite the exquisite care with which some of these syntheses have been performed, questions still remain with respect to TASP preparations as homogeneous molecular species of defined covalent composition. A convenient, direct general preparation of these molecules in unambiguous fashion would have great utility.
Recently, M. Schnolzer introduced the chemoselective ligation of unprotected peptide segments as a route to the total chemical synthesis of protein analogs of native (i.e. linear) topology (Science, 1992, vol. 256, pages 221-225). This approach uses unique, mutually reactive functionalities, one type on each segment, to covalently assemble long chain molecules from completely unprotected peptide segments. In this way, maximal advantage is taken of our ability to synthesize, handle, purify, and characterize unprotected peptides. Solubility problems are reduced and the target molecule is produced directly in the final unprotected form.